Lignin is the major structural component of secondarily thickened plant cell walls. It is a complex polymer of hydroxylated and methoxylated phenylpropane units, linked via oxidative coupling that is probably catalyzed by both peroxidases and laccases (Boudet, et al. 1995. “Tansley review No. 80: Biochemistry and molecular biology of lignification,” New Phytologist 129:203-236). Lignin imparts mechanical strength to stems and trunks, and hydrophobicity to water-conducting vascular elements. Although the basic enzymology of lignin biosynthesis is reasonably well understood, the regulatory steps in lignin biosynthesis and deposition remain to be defined (Davin, L. B. and Lewis, N. G. 1992. “Phenylpropanoid metabolism: biosynthesis of monolignols, lignans and neolignans, lignins and suberins,” Rec Adv Phytochem 26:325-375).
There is considerable interest in the potential for genetic manipulation of lignin levels and/or composition to help improve digestibility of forages and pulping properties of trees (Dixon, et al. 1994. “Genetic manipulation of lignin and phenylpropanoid compounds involved in interactions with microorganisms,” Rec Adv Phytochem 28:153178; Tabe, et al. 1993. “Genetic engineering of grain and pasture legumes for improved nutritive value,” Genetica 90:181-200; Whetten, R. and Sederoff, R. 1991. “Genetic engineering of wood,” Forest Ecology and Management 43:301-316). Small decreases in lignin content have been reported to positively impact the digestibility of forages (Casler, M. D. 1987. “In vitro digestibility of dry matter and cell wall constituents of smooth bromegrass forage,” Crop Sci 27:931-934). By improving the digestibility of forages, higher profitability can be achieved in cattle and related industries. In forestry, chemical treatments necessary for the removal of lignin from trees are costly and potentially polluting.
Lignins contain three major monomer species, termed p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S), produced by reduction of CoA thioesters of coumaric, ferulic and sinapic acids, respectively (see FIG. 1). In angiosperms, guaiacyl and syringyl units predominate, and the S/G ratio affects the physical properties of the lignin. The S and G units are linked through five different dimer bonding patterns (Davin, L. B. and Lewis, N. G. 1992. Rec Adv Phytochem 26:325-375). The mechanisms that determine the relative proportions of these linkage types in a particular lignin polymer are currently unknown. Furthermore, there is considerable debate as to whether lignin composition and structure are tightly controlled, or are flexible depending upon monomer availability (Lewis, N. G. 1999. “A 20th century roller coaster ride: a short account of lignification,” Current Opinion in Plant Biology 2:153-162; Sederoff, et al. 1999, “Unexpected variation in lignin,” Current Opinion in Plant Biology 2:145-152).
Lignin levels increase with progressive maturity in stems of forage crops, including legumes such as alfalfa (Jung, H. G. and Vogel, K. P. 1986. “Influence of lignin on digestibility of forage cell wall material,” J Anim Sci 62:1703-1712) and in grasses such as tall fescue (Buxton, D. R. and Russell, J. R. 1988. “Lignin constituents and cell wall digestibility of grass and legume stems,” Crop Sci 28:553-558). In addition, lignin composition changes with advanced maturity towards a progressively higher S/G ratio (Buxton, D. R. and Russell, J. R. 1988. Crop Sci 28:553-558). Both lignin concentration (Albrecht, et al. 1987. “Cell-wall composition and digestibility of alfalfa stems and leaves,” Crop Sci 27:735-741; Casler, M. D. 1987. Crop Sci 27:931-934; Jung, H. G. and Vogel, K. P. 1986. J Anim Sci 62:1703-1712) and lignin methoxyl content, reflecting increased S/G ratio (Sewalt, et al. 1996. “Lignin impact on fiber degradation. 1. Quinone methide intermediates formed from lignin during in vitro fermentation of corn stover,” J Sci Food Agric 71:195-203), have been reported to negatively correlate with forage digestibility for ruminant animals. Although a number of studies have linked decreased forage digestibility to increased S/G ratio as a function of increased maturity (Buxton, D. R. and Russell, J. R. 1988. Crop Sci 28:553-558; Grabber, et al. 1992. “Digestion kinetics of parenchyma and sclerenchyma cell walls isolated from orchardgrass and switchgrass,” Crop Sci 32: 806-810), other studies have questioned the effect of lignin composition on digestibility (Grabber, et al. 1997. “p-hydroxyphenyl, guaiacyl, and syringyl lignins have similar inhibitory effects on wall degradability,” J Agric Food Chem 45:2530-2532). Further, the hardwood gymnosperm lignins are highly condensed, essentially lacking S residues, and this makes them less amenable to chemical pulping, in apparent contradiction to the concept that reducing S/G ratio would be beneficial for forage digestibility. The reported lack of agreement in the relationship of lignin composition to forage digestibility and chemical pulping is partly due to the fact that the studies to date either have been in vitro, or have compared plant materials at different developmental stages, different varieties or even different species. Therefore, the development of isogenic lines that can be directly compared to reveal the effects of altered S/G ratio on forage digestibility would be beneficial.
The formation of the G and S units of lignin requires the activity of O-methyl-transferase enzymes. In angiosperms, the caffeic acid 3-O-methyltransferase (COMT) of lignin biosynthesis was originally described as being bifunctional, converting caffeic acid to ferulic acid and converting 5-hydroxyferulic acid to sinapic acid (Davin, L. B. and Lewis, N. G. 1992. Rec Adv Phytochem 26:325-375), as shown in FIG. 1. Methylation of the caffeate moiety also occurs at the level of the CoA thioester, catalyzed by caffeoyl CoA 3-O-methyltransferase (CCOMT) (Pakusch, et al., 1989, “S-adenosyl-L-methionine: trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures,” Arch Biochem Biophys 271:488-494). The involvement of the CCOMT enzyme in a parallel pathway to lignin monomer formation has been proposed (Ye, et al. 1994. “An alternative methylation pathway in lignin biosynthesis in Zinnia,” Plant Cell 6:1427-1439; Zhong, et al. 1998. “Dual methylation pathways in lignin biosynthesis,” Plant Cell 10:2033-2045). In vivo labeling studies in Magnolia kobus have shown that the methylation status of lignin monomers can also be determined at the level of the aldehyde or alcohol (Chen, et al. 1999. “Evidence for a novel biosynthetic pathway that regulates the ratio of syringyl to guaiacyl residues in lignin in the differentiating xylem of Magnolia kobus D C,” Planta 207:597-603). This is supported by the observation that the enzyme designated as ferulate 5-hydroxylase has a higher affinity for feruloyl aldehyde than for ferulic acid, at least in sweet gum (Osakabe, et al. 1999. “Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms,” Proc Natl Acad Sci USA 96:8955-8960) and Arabidopsis (Humphreys, et al. 1999. “New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase,” Proc Natl Acad Sci USA 96:10045-10050). Furthermore, 5-hydroxyconiferyl aldehyde has recently been shown to be a good substrate for COMT from various tree species (Li, et al. 2000. “5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms,” J Biol Chem 275:6537-6545). It has been reported that the inhibitory effect of 5-hydroxyconiferyl aldehyde on methylation of caffeate by COMT might prevent COMT from carrying out the first methylation step in the biosynthesis of S lignin (Li, et al. 2000. J Biol Chem 275:6537-6545). Thus, although studies of enzyme substrate specificities in vitro suggest that lignin monomers can be formed via the operation of a complex metabolic grid, involving O-methylation at multiple stages as shown in FIG. 1, whether this occurs in vivo has yet to be determined.
Several studies have addressed the properties of the O-methyltransferases involved in lignin biosynthesis in the world's major forage legume, alfalfa (Medicago sativa L.) (Gowri, et al. 1991. “Stress responses in alfalfa (Medicago sativa L.) X. Molecular cloning and expression of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase, a key enzyme of lignin biosynthesis,” Plant Physiol 97:7-14; Inoue, et al. 1998. “Developmental expression and substrate specificities of alfalfa caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in relation to lignification,” Plant Physiol 117:761-770; Kersey, et al. 1999. “Immunolocalization of two lignin O-methyltransferases in stems of alfalfa (Medicago sativa L.),” Protoplasma 209:46-57). COMT from alfalfa expressed in E. coli shows preference (approximately 2:1) for 5-hydroxyferulic acid over caffeic acid, whereas CCOMT shows a similar degree of preference for caffeoyl CoA compared to 5-hydroxyferuolyl CoA (Inoue, et al. 1998. Plant Physiol 117:761-770). These studies suggest, but do not prove, that COMT may be involved preferentially in the formation of S lignin in alfalfa, and CCOMT in the formation of G lignin.
The substrate preference of COMT in crude alfalfa stem extracts changes with increasing internode maturity, in a manner consistent with the increase in lignin methoxyl group content with increasing maturity (Inoue, et al. 1998. Plant Physiol 117:761-770; Inoue, et al. 2000. “Substrate preferences of caffeic acid/5-hydroxyferulic acid 3-O-methyltransferases in developing stems of alfalfa (Medicago sativa L.),” Arch Biochem Biophys 375:175-182). Thus, in young internodes, the activity shows a preference for caffeic acid over 5-hydroxyferulic acid, whereas the opposite is true in the older internodes. An O-methyltransferase with preference for caffeic acid (COMT II) has recently been separated from the previously characterized COMT, and does not react with antisera recognizing the products of the previously characterized alfalfa COMT or CCOMT genes. This enzyme is most active against caffeic acid, for which it has a very low Km value (approximately 40-fold lower than lignification-associated COMT), but also methylates 5-hydroxyferulic acid, caffeoyl CoA, 5-hydroxyferuolyl CoA, quercetin and catechol (Inoue, et al. 2000. Arch Biochem Biophys 375:175-182). It is only present in young internodes and has disappeared by the fifth internode.
Tissue print hybridization analysis indicates that both COMT and CCOMT transcripts are localized to developing xylem elements in alfalfa stems, whereas CCOMT transcripts are also found in phloem (Inoue, et al. 1998. Plant Physiology 117:761-770). Immunolocalization studies at the light and electron microscope levels demonstrated expression of both COMT and CCOMT in the cytoplasm of alfalfa xylem parenchyma cells (Kersey, et al. 1999 Protoplasma 209:46-57). The presence of both enzymes in the same cells is consistent with the “metabolic grid” hypothesis for lignin monomer formation.
There have been several reports on the effects of down-regulation of COMT activity on lignin content and composition in transgenic tobacco and poplar (Ni, et al. 1994. “Reduced lignin in transgenic plants containing an engineered caffeic acid O-methyltransferase antisense gene,” Transgenic Res 3:120-126; Atanassova, et al. 1995. “Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation,” Plant J 8:465-477; Van Doorsselaere, et al. 1995. “A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity,” Plant J 8:855-864; Zhong, et al. 1998. Plant Cell 10:2033-2045). The results of these studies have been somewhat contradictory, possibly due to unspecified differences in tissue maturity, use of homologous versus heterologous transgenes, and use of different methods for lignin analysis. However, in cases where COMT has been reduced to levels below approximately 20% of wild-type by expression of a homologous transgene, a strong reduction in S/G ratio is accompanied by no apparent change in lignin content (Atanassova, et al. 1995. Plant J 8:465-477; Van Doorsselaere, et al. 1995. Plant J 8:855-864). In tobacco, down-regulation of CCOMT leads to a corresponding decrease in Klason lignin levels accompanied by decreases in the absolute levels of both S and G units (Zhong, et al. 1998. Plant Cell 10:2033-2045).
Most studies on genetic modification of lignin biosynthesis in transgenic plants have utilized the cauliflower mosaic virus 35S promoter to drive expression of sense or antisense lignification-associated genes (Halpin, et al. 1994. “Manipulation of lignin quality by down-regulation of cinnamyl alcohol dehydrogenase,” Plant J 6:339-350; Ni, et al. 1994. Transgenic Res 3:120-126; Atanassova, et al. 1995. Plant J 8:465-477; Van Doorsselaere, et al. 1995. Plant J 8:855-864; Piquemal, et al. 1998. “Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants,” Plant J 13:71-83; Zhong, et al. 1998. Plant Cell 10:2033-2045; Baucher, et al. 1999, “Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility,” Plant Mol Biol 39:437-447). However, more effective down-regulation may be obtained by driving expression of the transgene by a vascular-tissue specific promoter. For example, modification of lignin composition by overexpression of ferulate 5-hydroxylase in transgenic Arabidopsis was more effective if the transgene was driven by the lignification-associated Arabidopsis cinnamate 4-hydroxylase promoter than by the constitutive 35S promoter (Meyer, et al. 1998. “Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis,” Proc Natl Acad Sci USA 95:6619-6623).
To date, there have been very few published reports on the genetic modification of lignin in forage crops, and most studies having concentrated on model systems such as Arabidopsis and tobacco, or tree species such a poplar. In one study, down-regulation of cinnamyl alcohol dehydrogenase, an enzyme later in the monolignol pathway than COMT or CCOMT, led to a small but significant improvement in in vitro dry matter digestibility in transgenic alfalfa (Baucher, et al. 1999. Plant Mol Biol 39:437-447). U.S. Pat. No. 5,451,514 discloses a method of altering the content or composition of lignin in a plant by stably incorporating into the genome of the plant a recombinant DNA encoding an mRNA having sequence similarity to cinnamyl alcohol dehydrogenase. U.S. Pat. No. 5,850,020 discloses a method for modulating lignin content or composition by transforming a plant cell with a DNA construct with at least one open reading frame coding for a functional portion of one of several enzymes isolated from Pinus radiata (pine) or a sequence having 99% homology to the isolated gene: cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyltransferase (OMT), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL), and peroxidase (POX). U.S. Pat. No. 5,922,928 discloses a method of transforming and regenerating Populus species to alter the lignin content and composition using an O-methyltransferase gene. The question of how altering S/G ratio might affect digestibility of forage species is still unanswered.
It has now been found that transformation of plants with the lignin biosynthetic enzyme genes COMT or CCOMT in either a sense or antisense orientation under a lignification-associated tissue specific promoter, leading to the down-regulation of the corresponding homologous gene as well as reduced lignin content and modified lignin composition in the transgenic plants, results in significant improvements in forage digestibility, particularly in the case of CCOMT down-regulation.